Neutralising antibody responses against SARS-CoV-2 Omicron BA.4/5 and wild-type virus in patients with inflammatory bowel disease following three doses of COVID-19 vaccine (VIP): a prospective, multicentre, cohort study

Summary Background Patients with inflammatory bowel disease (IBD) receiving anti-TNF and JAK-inhibitor therapy have attenuated responses to COVID-19 vaccination. We aimed to determine how IBD treatments affect neutralising antibody responses against the Omicron BA.4/5 variant. Methods In this multicentre cohort study, we prospectively recruited 340 adults (69 healthy controls and 271 IBD) at nine UK hospitals between May 28, 2021 and March 29, 2022. The IBD study population was established (>12 weeks therapy) on either thiopurine (n = 63), infliximab (n = 45), thiopurine and infliximab combination therapy (n = 48), ustekinumab (n = 45), vedolizumab (n = 46) or tofacitinib (n = 24). Patients were excluded if they were being treated with any other immunosuppressive therapies. Participants had two doses of either ChAdOx1 nCoV-19 or BNT162b2 vaccines, followed by a third dose of either BNT162b2 or mRNA1273. Pseudo-neutralisation assays against SARS-CoV-2 wild-type and BA.4/5 were performed. The half maximal inhibitory concentration (NT50) of participant sera was calculated. The primary outcome was anti-SARS-CoV-2 neutralising response against wild-type virus and Omicron BA.4/5 variant after the second and third doses of anti-SARS-CoV-2 vaccine, stratified by immunosuppressive therapy, adjusting for prior infection, vaccine type, age, and interval between vaccination and blood collection. This study is registered with ISRCTN (No. 13495664). Findings Both heterologous (first two doses adenovirus vaccine, third dose mRNA vaccine) and homologous (three doses mRNA vaccine) vaccination strategies significantly increased neutralising titres against both wild-type SARS-CoV-2 virus and the Omicron BA.4/5 variant in healthy participants and patients with IBD. Antibody titres against BA.4/5 were significantly lower than antibodies against wild-type virus in both healthy participants and patients with IBD (p < 0.0001). Multivariable models demonstrated that neutralising antibodies against BA.4/5 after three doses of vaccine were significantly lower in patients with IBD on infliximab (Geometric Mean Ratio (GMR) 0.19 [0.10, 0.36], p < 0.0001), infliximab and thiopurine combination (GMR 0.25 [0.13, 0.49], p < 0.0001) or tofacitinib (GMR 0.43 [0.20, 0.91], p = 0.028), but not in patients on thiopurine monotherapy, ustekinumab, or vedolizumab. Breakthrough infection was associated with lower neutralising antibodies against wild-type (p = 0.037) and BA.4/5 (p = 0.045). Interpretation A third dose of a COVID-19 mRNA vaccine based on the wild-type spike glycoprotein significantly boosts neutralising antibody titres in patients with IBD. However, responses are lower against the Omicron variant BA.4/5, particularly in patients taking anti-TNF and JAK-inhibitor therapy. Breakthrough infections are associated with lower neutralising antibodies and immunosuppressed patients with IBD may receive additional benefit from bivalent vaccine boosters which target Omicron variants. Funding 10.13039/100004319Pfizer.


Introduction
2][3][4][5] However, since vaccines are developed mainly from trials recruiting healthy people, data on vaccine efficacy in immunosuppressed populations, including patients with inflammatory bowel disease (IBD) taking immunosuppressive drugs, are lacking.The emergence of new variants, particularly more transmissible strains, such as the Omicron variants, has resulted in high infection rates in unvaccinated and vaccinated individuals. 6Specifically, due to the L452R and F486V mutations in the spike protein, the BA.4/5 variant displayed reduced neutralisation by the serum from individuals vaccinated with three doses of vaccine compared with precedent Omicron variants. 7Concerns about more transmissible variants are especially pertinent for immunosuppressed individuals, who are more prone to infection, viral persistence and COVID-19 disease or have impaired immune responses to vaccination. 8Viral infection and persistence constitute a threat, both to patient health and pandemic control, with the risk of disease, onward transmission and evolution of new variants of concern (VOC).0][11][12] Anti-TNF treatment is also associated with accelerated loss of circulating vaccine-induced antibodies and anti-TNF treated patients are more likely to develop breakthrough infections after two doses of vaccine. 8,12,13n some countries, including the UK, patients treated with immunosuppressant drugs, including patients with IBD are prioritised to receive a third primary and booster vaccine dose. 14The British Society of Gastroenterology advises that a third dose of a SARS-CoV-2 vaccine should be offered no earlier than 8 weeks after the second dose to all patients with inflammatory bowel disease who are 12 years and over and who are receiving any immunosuppressive treatment.There is no antibody testing prior to vaccination and few results or outcomes have been reported following the third dose of vaccine in patients with IBD.In addition, heterologous vaccination regimens (e.g. two doses of adenovirus vector vaccine followed by one dose of mRNA vaccine) have been used in many countries and were reported to be effective in healthy individuals. 15Given the continuing emergence of Omicron variants, further studies are needed to understand how key immunosuppressive drug regimens impact vaccine-induced immunogenicity against the more transmissible variant.This study aimed to evaluate the functional neutralising responses directed against BA.4/5 in an immunosuppressed population of patients with IBD.

Study design and participants
VIP (SARS-CoV-2 Vaccination Immunogenicity in Immunosuppressed inflammatory bowel disease Patients) is a UK multi-centre prospective observational study aimed to evaluate the immunogenicity of COVID-19 vaccination in patients with IBD on six different immunosuppressive treatment regimens (infliximab, thiopurine, infliximab and thiopurine combination therapy, ustekinumab, vedolizumab or tofacitinib).This study adheres to the STROBE guidelines.Participant recruitment, inclusion and exclusion criteria have been described previously. 9Blood was collected from participants 53-92 days after the second vaccine dose and 28-49 days after the third vaccine dose (Fig. 1).Participants either received homologous (3 doses of mRNA vaccine) or heterologous (2 doses of adenovirus vector followed by one dose of mRNA vaccine) vaccination schedules.
Participants were included after providing informed, written consent.The Wales Research Ethics Committee 5 approved the study (REC reference 21/WA/0105) in March, 2021.The study was registered with the ISRCTN (No: 13495664) registry.

Anti-SARS-CoV-2 antibody electrochemiluminescence assay
The Roche Elecsys anti-SARS-CoV-2 (N) immunoassay is a sandwich electrochemiluminescence immunoassay that employs a recombinant protein of the nucleocapsid antigen for the determination of antibodies against SARS-CoV-2 infection.The manufacturer reports clinical sensitivity and specificity of 99.5% and 99.8%,

Research in context
Evidence before this study We searched PubMed and Embase, without language restrictions, for studies published between Jan 1, 2000 and Oct 31, 2022, investigating humoral responses to vaccination in immunosuppressed individuals.We used the search terms ("vaccine" OR "vaccination") AND ("immunosuppression" OR "immunosuppressive" OR "immunomodulator" OR "thiopurine" OR "azathioprine" OR "biologic" OR "tumour necrosis factor" OR "infliximab" OR "ustekinumab" OR "antiintegrin" OR "vedolizumab" OR "JAK inhibitor" OR "tofacitinib") AND ("antibody" OR "humoral" OR "immune response") OR ("Omicron").We have previously shown that third doses of COVID-19 vaccines boost serological responses in patients with inflammatory bowel disease (IBD) receiving six different commonly used immunosuppressive treatment regimens.However, COVID-19 vaccine-induced antibody responses are diminished relative to healthy controls in patients with IBD taking anti-TNF and JAK-inhibitor therapies, but not anti-integrin or thiopurine monotherapy, following two and three vaccine doses.Breakthrough infection is more common in patients with IBD receiving the anti-TNF therapy infliximab compared with the gut-selective anti-integrin therapy vedolizumab.We have recently shown that neutralising antibody responses against the Omicron BA.1 variant of concern are reduced in immunosuppressed patients with IBD in comparison to neutralising responses against the wild-type virus.However, there is currently scarce data on neutralising responses against the Omicron BA.4/5 variant and the related risk of infection.

Added value of this study
To our knowledge, this is the first study to evaluate neutralising antibodies against the Omicron BA.4/5 variant following three doses of COVID-19 vaccine in patients receiving different immunosuppressive treatments used in IBD.We show that, although all groups had a significant boost in vaccine-induced neutralising antibody responses after a third dose, levels were significantly reduced in those patients treated with infliximab or tofacitinib.Neutralising responses against BA.4/5 in patients receiving infliximab and tofacitinib were more than one order of magnitude lower than against wild-type virus and lower neutralising antibodies against BA.4/5 were associated with a higher risk of breakthrough infection.

Implications of all the available evidence
Our data show that a third dose of an originator COVID-19 vaccine targeting the wild-type virus spike protein boosts neutralising antibodies against the Omicron BA.4/5 variant in immunosuppressed patients with IBD, but responses against BA.4/5 are significantly lower irrespective of immunosuppressive treatment.Combined with evidence that previous SARS-CoV-2 infection further augments humoral responses to vaccination, these results support the rollout of booster doses in immunosuppressed patients with IBD.In the context of emerging variants of concern, and evidence that patients treated with anti-TNF are at higher risk of breakthrough infection, our data also support the prioritisation of future booster dosing to those with diminished responses to vaccination, including patients taking anti-TNF or tofacitinib.
respectively, >14 days after PCR-confirmed COVID-19 using a cut-off index (COI) of 1.It is reported that anti-N antibody responses following SARS-CoV-2 natural infection are impaired in patients treated with immunosuppressant drugs such as infliximab. 8,16Results showed that a threshold of 0.12 times the cut-off index provides 100% specificity for determining prior SARS-CoV-2 infection. 12Therefore, in the current study, anti-N greater than or equal to 0.12 was deemed to indicate prior SARS-CoV-2 infection.

Pseudo neutralisation assay
The SARS-CoV-2 neutralisation assays were conducted using pseudo-typed viruses (PSV).Pseudo-typed SARS-CoV-2 lentiviruses were produced in HEK293T cells using a SARS-CoV-2 spike plasmid (wild-type strain or BA.4/5), HIV-1 gag-pol plasmid and a firefly luciferase reporter. 17Participant sera were serially diluted and incubated with PSV viral supernatant for 1 h.HEK293T-ACE2 cells were then co-incubated with the sera and PSV for 72 h before measurement of the luciferase activity using the Bright-Glo Luciferase assay system (Promega, Madison, WI).NT50 neutralisation titres were calculated as the dilution at which relative luminescence was reduced by 50% compared with control.

Outcome measures
Our primary outcome was anti-SARS-CoV-2 neutralising response against wild-type virus and the Omicron BA.4/5 variant after the second and third doses of anti-SARS-CoV-2 vaccine, stratified by baseline immunosuppressive therapy, adjusting for prior infection, vaccine type, age, and the interval between vaccination and blood sampling.
The secondary outcome was risk of breakthrough infection after two doses of vaccination.SARS-CoV-2 infection was defined by participants who reported a PCR or lateral flow test confirming SARS-CoV-2 infection and/or a concentration of Roche Elecsys anti-SARS-CoV-2 nucleocapsid immunoassay nucleocapsid antibodies in the serum above 0.11 U/mL. 9,12The data collection range was between 28th May 2021 and 29th March 2022.

Variables
Demographics were recorded as variables: age, gender, ethnicity, comorbidities, height and weight, smoking status, postcode, IBD disease activity (defined by patientreported outcomes [PRO2]), 18,19 SARS-CoV-2 symptoms aligned to the COVID-19 symptoms study (symptoms, previous testing and hospital admissions for COVID-19), SARS-CoV-2 test date and results, vaccine schedules (type and date of each vaccination) and date of blood collection.Data were entered electronically into a purpose-designed REDCap database hosted at the Royal Devon University Healthcare NHS Foundation Trust. 20articipants without access to the internet or electronic devices completed their questionnaires on paper case record forms that were subsequently entered by local research teams.

Statistical analysis
A statistical analysis plan was approved by the Study Management.Analyses were undertaken using R 4.1.0(R Foundation for Statistical Computing, Vienna, Austria).p values < 0.05 with two-tailed tests were considered significant.Antibody concentrations are reported as geometric mean and standard deviation.Other continuous data are reported as a median and interquartile range, and discrete data as numbers and percentages, unless otherwise stated.The comparison of breakthrough infection rates in patients with IBD above and below NT50 cut-off was performed with Fisher's exact test.To determine the NT50 cut-off value, we initially consulted relevant literature to understand commonly used cut-off values.However, due to methodological differences across studies, these values were not directly comparable.Therefore, we conducted our own data analysis, comparing breakthrough infection rates at various NT50 levels.This allowed us to identify a cut-off value that was most effective in differentiating breakthrough infection rates in our specific study population.
Multivariable linear regression models were used to identify factors independently associated with neutralising antibody concentrations.These variables were initially tested in the regression models: age, gender, ethnicity, body mass index, height, weight, smoking, IBD subtype, first two doses of vaccine type (mRNA vs adenovirus vector vaccine), homologous (3 doses of mRNA) or heterologous (2 doses of adenovirus vector followed by 1 dose of mRNA vaccine) vaccination schedules, interval days between the second dose of vaccine and first blood sampling, interval days between the third dose of vaccine and second blood sampling, interval days between the first and second dose of vaccination, and interval days between the second and third dose of vaccination.We used backward stepwise regression and calculated the Akaike Information Criterion (AIC), which takes into account both the goodness of fit of the model and the likelihood of overfitting.In this approach, variables are iteratively removed from the model in a way that results in the largest decrease in AIC.Based on their clinical significance, and for consistency across all models including those after two and three vaccine doses and against both the wild-type and Omicron variants, we included the following variables in the final models: age, prior infection, interval in weeks from vaccination to blood sampling, and mRNA vaccination (or homologous vaccination for the third vaccine dose).In the regression model for the third dose, we also included NT50 after two vaccine doses as a predictor, since it could influence the NT50 after three doses.Results are presented after exponentiation so that the model's coefficients correspond to the geometric mean ratio associated with each covariate.The linearity, homogeneity of variance, collinearity, influential observations, and normality of residuals of each multivariate model were assessed using the performance package in R. Linearity assumption for key continuous variables including age, NT50 after second dose and time from second or third dose to sampling were also assessed individually with partial regression plots using the car package in R.
We also performed sensitivity analyses to investigate how IBD affect the neutralising antibodies.We excluded healthy controls and performed multivariable regression analysis to verify the effect of IBD drugs on neutralising antibodies.Since vaccination, neutralising antibodies and prior infection are interrelated, we also carried out multivariable regression analysis excluding participants with prior infection.

Role of the funding source
The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.ZL, JLA and NP had access to dataset and had final responsibility for the decision to submit for publication.
To evaluate vaccine-induced humoral responses we employed a pseudo neutralisation assay against the SARS-CoV-2 wild-type (WT) and Omicron BA.4/5 variant.After two or three doses of vaccine, 50% neutralisation titres (NT50) against Omicron BA.4/5 were significantly lower compared to against WT both in healthy controls and all six IBD patient groups (p values < 0.0001 comparing NT50 against WT and BA.4/ 5 in each group.Fig. 2 effect of a third vaccine dose compared to two doses of vaccine.Reassuringly, neutralising titres against WT and BA.4/5 significantly increased after a third dose of vaccine compared to titres after a second dose of vaccine in all treatment groups (Supplementary Fig. S1 p < 0.0001 comparing NT50 after 2 and 3 vaccine doses in each group).We then plotted the NT50 between healthy controls and patients with IBD with different treatments.Patients treated with infliximab, infliximab plus thiopurine combination therapy, or tofacitinib showed lower NT50 against WT and BA.4/5 both after 2 and 3 vaccine doses compared to healthy controls (Fig. 3).27 patients did not generate an NT50 against the BA.4/5 variant after two doses of vaccine, 21 of whom received infliximab treatment, comprising 30% patients of all patients treated with infliximab (Fig. 3A).After 3 doses of vaccine, three patients treated with infliximab still did not generate an NT50 against BA.4/5 (Fig. 3B).
We performed multivariable linear regression to determine whether other variables impacted vaccineinduced immune responses.We first carried out model diagnostic checks to confirm the linear regression models perform well (Supplementary Figs.S2-S5).The model-predicted lines match the observed data in the posterior predictive check.Both linearity and homogeneity of variance checks show nearly flat and horizontal reference lines, confirming their assumptions are met.All points are inside the contour lines in the influential observations check, indicating no overly influential observations.The collinearity assessment indicates that the variance inflation factor (VIF) for all models falls below 2, signifying that multicollinearity is unlikely to be an issue.The normality of residuals check confirms that residuals are approximately normally distributed.Linearity assumption for key continuous variables such as age, NT50 after second dose and time from second or third dose to sampling were assessed by partial regression plots (Supplementary Figs.S6 and  S7), which demonstrated that the linearity assumption is likely met.Overall, the models are well-specified and fit the data well.
In the regression results, lower neutralising antibody titres against the WT virus and the BA.4/5 variant were observed in patients with IBD established on infliximab monotherapy (all comparisons p < 0.01), infliximab and thiopurine combination therapy (all comparisons p < 0.01) or tofacitinib (all comparisons p < 0.05), after two or three doses of COVID-19 vaccine (Figs. 4 and 5).Prior infection was independently associated with higher NT50 (all comparisons p < 0.001).Older age was associated with lower NT50 against WT after two doses (p = 0.022), against BA.doses of mRNA vaccine induced higher titres of neutralising antibodies than two doses of adenovirus vector vaccine (both p < 0.01).All patients received mRNA vaccine as the third dose, and there was no significant difference in the neutralising titres after three doses between homologous and heterologous booster schedules (both p > 0.05).
To investigate variations in neutralising antibody levels among patients with IBD treated with different drugs, we conducted a multivariable regression analysis excluding healthy controls (Supplementary Figs.S8 and  S9).In light of both our study and the CLARITY study [21] indicating that vedolizumab does not impair antibody responses, we used vedolizumab-treated patients as the reference group.The results, which excluded healthy controls, were consistent with our original findings that included healthy controls, demonstrating that treatment with infliximab, the combination of infliximab and thiopurine, and tofacitinib resulted in lower neutralising antibody responses.
We also performed a sub-analysis excluding participants with prior infections (Supplementary Figs.S10  and S11).The neutralising antibodies were lower in patients treated with infliximab, the combination of infliximab and thiopurine, and tofacitinib after two vaccine doses (all p < 0.05).As time went on, more people contracted the infection, so more participants were excluded after the third vaccine dose.With a smaller sample size, some comparisons became nonsignificant, but the trend remained the same.After three doses of vaccination, lower neutralising antibodies were found in patients treated with infliximab (p = 0.10 for wild-type, p = 0.012 for BA.4/5), a combination of infliximab and thiopurine (p = 0.0039 for wild-type, p = 0.0011 for BA.4/5), and tofacitinib (p = 0.0027 for wild-type, p = 0.13 for BA.4/5).
Next, we investigated the breakthrough infection rate in this cohort, which was 17% in either healthy controls or patients with IBD.Then we tested the correlation of different thresholds of neutralising titres with the breakthrough infection rate (Fig. 6A).Patients with IBD with an NT50 < 500 against the WT virus after two vaccine doses had a 1.88-fold increased risk of infection compared to patients with IBD with NT50 > 500 (p = 0.037).Patients with IBD with an NT50 < 17 against BA.4/5 after two vaccine doses had a 1.96-fold increased risk of infection compared to patients with IBD with NT50 > 17 (p = 0.045).We also stratified the distribution of NT50 in breakthrough cases by immunosuppressive treatments and compared it to the distribution in uninfected cases (Fig. 6B and C).No statistically significant difference among the groups was identified which may be attributable to small sample sizes after stratification.There were fewer healthy controls with NT50 under the cut-off and no significant associations between breakthrough infection and NT50 (above or under cut-off).

Discussion
In this study, we have shown that neutralising antibody responses against SARS-CoV-2 wild-type and BA.evolving variants, antibodies elicited against the wildtype spike will inevitably have reduced binding and neutralising activity against these novel variants.Since many mutations exist in the Omicron spike protein, this might lead to a significant escape from immune protection elicited by COVID-19 vaccine designed against SARS-CoV-2 wild-type virus. 21ur data also highlight some other interesting findings relevant to the care of patients with IBD.A third dose of the mRNA vaccine significantly improved responses in patients who had previously received their first two doses with either mRNA or the ChAdOx1 nCoV-19 vaccine, showing that both homologous and heterologous boosting with mRNA vaccines is effective in patients with IBD. 22,23Prior infection was independently associated with higher vaccine-induced neutralising antibody responses, even after a third dose of vaccine, consistent with the likelihood that further vaccine boosters will continue to incrementally increase neutralising antibody responses, which may be especially important in patients treated with anti-TNF drugs or tofacitinib, who have lower circulating antibody levels, particularly against emerging VOCs.Lower NT50 against the BA.4/5 variant was associated with higher risk of breakthrough infection, suggesting further booster, especially the recently available bivalent vaccine which was designed based on Omicron variants should be rolled out to protect people from infection or severe diseases. 24his study did not explore the immunological mechanisms for reduced vaccine-induced antibody responses observed in infliximab and tofacitinib.[34]  Consistent with our findings, CLARITY-IBD found that patients with IBD treated with infliximab had lower anti-S1 or neutralising antibodies against the SARS-CoV-2 wild-type and Omicron variants compared to those treated with vedolizumab after 2 or 3 vaccine doses. 8,12,16,35Although this was associated with an increased risk of breakthrough infection in infliximab recipients, COVID symptoms were mild, and severe disease, including hospitalisations and deaths, was reassuringly still uncommon. 87][38][39] Interestingly, two independent studies have observed augmented antigen-specific T-cell responses in anti-TNF treated patients with IBD following 2 doses of SARS-CoV-2 vaccine doses, 36,40 which may be important in providing anti-viral activity despite attenuated antibody responses, and may partly explain why outcomes do not appear to be worse in anti-TNF recipients following infection. 8ur study has important strengths.We have taken a unique approach to evaluating antibody responses directed against the dominating variant BA.4/5 in late 2022.We have also harnessed functional neutralisation assays, rather than anti-S1 serology assays employed in most other IBD studies.We have also actively recruited patients established on the main IBD drug regimens to get a broad view of the impact of different immunosuppressive mechanisms of action on vaccine-induced immunogenicity.We also prospectively recruited a population of healthy, non-IBD controls as a critical comparison.However, we acknowledge the limitations of this study.Firstly, the sample size for some drug groups, most notably tofacitinib, was small, which might limit the robustness of our findings, although our observations were highly statistically significant.Secondly, we do not report anti-viral T-cell immune responses in the current work, although we found that Tcell responses were similar in all treatment groups, except for reduced T-cell responses in patients treated with tofacitinib. 10Thirdly, the prior infection history was self-reported by participants who had been confirmed with the SARS-CoV-2 PCR test, or lateral flow testing, which may be susceptible to recall bias.Finally, although our study was consistent with a signal for increased risk of breakthrough infection in patients with IBD with lower titres of neutralising antibodies, the study was underpowered to answer this question definitely, and the results should be regarded with caution.

A B
In summary, we have shown that third dose of vaccination significantly increases neutralising antibody responses against the SARS-CoV-2 wild-type and Omicron BA.4/5 variants in both healthy people and immunosuppressed patients with IBD.With more mutations in the Spike protein and ease of immune escape, antibodies to BA.4/5 are lower compared to wild-type, indicating that as new variants and mutations emerge, further vaccination, especially with secondgeneration bivalent vaccines targeting Omicron, is warranted to raise the antibody levels against the virus, especially in patients treated with the anti-TNF drug infliximab and the JAK inhibitor tofacitinib.

BFig. 2 :
Fig. 2: NT50 against SARS-CoV-2 wild-type (WT) and BA.4/5 in patients with IBD treated with different immunosuppressive medications and healthy controls after two (A) or three (B) doses of vaccine.The crossbar represented the geometric mean and SD.Wilcoxon signed-rank test was performed to test the significance.Samples unable to inhibit half of the virus infection were plotted with the NT50 equal to 0.1.

Fig. 3 :
Fig. 3: NT50 in patients with IBD treated with different immunosuppressive medications and healthy controls against SARS-CoV-2 wild-type (WT) and BA.4/5 after two (A) or three (B) doses of vaccine.The crossbar represented the geometric mean and SD.Samples unable to inhibit half of the virus infection were plotted with the NT50 equal to 0.1.

Fig. 5 :
Fig. 5: Multivariable regression model showing the exponentiated coefficients of linear regression models of log-transformed NT50 after three doses stratified by study treatment group.A: NT50 against wild-type.B: NT50 against BA.4/5.The values represent the geometric mean ratios of NT50 associated with each variable.

Fig. 6 :
Fig.6: The correlation of breakthrough infection with NT50 after two vaccine doses in patients with IBD.A: The percentage of breakthrough infection above and below NT50 cut-off.Statistical difference was calculated with a Fisher's test.B: NT50 against wild-type after two doses of vaccine stratified by treatment and breakthrough infection.C: NT50 against BA.4/5 after two doses of vaccine stratified by treatment and breakthrough infection.The crossbar represented the geometric mean and SD.
, Table2).We next evaluated the Continuous variables were presented as median (IQR).Other variables were presented as percentages within each group.Kruskal-Wallis H test (for continuous variables) and Fisher's exact test (for categorical variables) were employed to test the significance.BMI: body mass index.Table 1: Demographics of the cohort in this study.

per decade) mRNA vaccine Prior infection Interval weeks: Dose 2 to sampling
Multivariable regression model, adjusted for age, prior infection, first two doses of vaccine type (mRNA vs adenovirus vector vaccine) and interval between second vaccine dose and blood sampling, showing the exponentiated coefficients of linear regression models of logtransformed NT50 after two doses stratified by study treatment group.A: NT50 against wild-type.B: NT50 against BA.4/5.The values represent the geometric mean ratios of NT50 associated with each variable.